Genetic traits and transmission of antimicrobial resistance characteristics of cephalosporin resistant Escherichia coli in tropical aquatic environments.

This study investigates the genetic traits and transmission mechanisms of cephalosporin-resistant Escherichia coli in tropical aquatic environments in Singapore. From 2016 to 2020, monthly samples were collected from wastewater treatment plants, marine niches, community sewage, beaches, reservoirs, aquaculture farms, and hospitals, yielding 557 isolates that were analyzed for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) using genomic methods. Findings reveal significant genotypic similarities between environmental and hospital-derived strains, particularly the pandemic E. coli ST131. Environmental strains exhibited high levels of intrinsic resistance mechanisms, including mutations in porins and efflux pumps, with key ARGs such as CMY-2 and NDM-9 predominantly carried by MGEs, which facilitate horizontal gene transfer. Notably, pathogenic EPEC and EHEC strains were detected in community sewage and aquaculture farms, posing substantial public health risks. This underscores the critical role of these environments as reservoirs for multidrug-resistant pathogens and emphasizes the interconnectedness of human activities and environmental health.

Sonodynamic inactivation of gram-negative and gram-positive bacteria in the presence of phenothiazine compounds toluidine blue and azurin A.

BACKGROUND: Sonodynamic antimicrobial chemotherapy (SACT) is an effective antimicrobial treatment that can avoid the production of drug-resistant bacteria. Design and development of new high-efficiency sonosensitizers play a key role in the practical application of SACT. METHODS: The bacteriostatic effects of two phenothiazine compounds, toluidine blue (TB) and azure A (AA) combined with ultrasonic (US) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were studied, and the sonodynamic antibacterial activities of TB and AA were compared. The reactive oxygen species (ROS) and the types of ROS produced in the sonodynamic system were detected and the sonodynamic mechanisms of TB and AA were proposed. RESULTS: The sonodynamic bacteriostasis mediated by TB and AA increased with the increasing concentration of sonosensitizer, the extension of sonication time and the increase of reaction temperature. The production of ROS was the main reason that TB and AA had excellent sonodynamic antibacterial performance. Singlet oxygen (1O2) and hydroxyl radical (•OH) were the main ROS types in the sonodynamic antibacterial system. The ROS produced by the combined action of AA and US was higher than that of TB. CONCLUSION: Both TB and AA displayed excellent sonodynamic antibacterial activities. Moreover, AA had a higher sonodynamic activity than TB. The electron donation effect and steric hindrance effect of the methyl group of phenothiazine parent nucleus of TB might be the cause of the decrease of its sonodynamic activity. These results would provide a valuable reference for the further study of phenothiazines sonosensitizers and their clinical application in SACT.

Various microbes used for the recovery of rare earth elements from mine wastewater.

Microbes used for the recovery of rare earth elements (REEs) from mining wastewater indicated traces of Escherichia coli (E. coli, 2149.6 µg/g), Bacillus sphaericus (1636.6 µg/g), Bacillus mycoides (1469.3 µg/g), and Bacillus cereus (1083.9 µg/g). Of these, E. coli showed an affinity for REEs than non-REEs (Mn and Zn). The amount of heavy REEs adsorbed (1511.1 µg/g) on E. coli was higher than light REEs (638.0 µg/g) due to the process of increasing adsorption with decreasing ionic radius. Additionally, E. coli demonstrated stability in the recovery of REEs from mining wastewater, as evidenced by 4 cycles. SEM-EDS, XPS and FTIR showed that REEs had a disruptive effect on cells, REEs absorbed and desorbed on the cell surface including ion exchange with ions such as Na+, ligand binding with functional groups like -NH2. Finally, the cost assessment confirmed the economically feasible of E. coli in recovery of REEs from mining wastewater.

Global Dynamics of Gastrointestinal Colonisations and Antimicrobial Resistance: Insights from International Travellers to Low- and Middle-Income Countries.

Gastrointestinal microorganism resistance and dissemination are increasing, partly due to international travel. This study investigated gastrointestinal colonisations and the acquisition of antimicrobial resistance (AMR) genes among international travellers moving between Spain and low- and middle-income countries (Peru and Ethiopia). We analysed 102 stool samples from 51 volunteers collected before and after travel, revealing significantly higher rates of colonisation by both bacteria and protists upon return. Diarrhoeagenic strains of E. coli were the most notable microorganism detected using RT-PCR with the Seegene Allplex™ Gastrointestinal Panel Assays. A striking prevalence of ß-lactamase resistance genes, particularly the TEM gene, was observed both before and after travel. No significant differences in AMR genes were found between the different locations. These findings highlight the need for rigorous surveillance and preventive strategies, as travel does not significantly impact AMR gene acquisition but does affect microbial colonisations. This study provides valuable insights into the intersection of gastrointestinal microorganism acquisition and AMR in international travellers, underscoring the need for targeted interventions and increased awareness.

Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia.

OBJECTIVES: Despite the critical importance of colistin as a last-resort antibiotic, limited studies have investigated colistin resistance in human infections in Cambodia. This study aimed to investigate the colistin resistance and its molecular determinants among Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing (CP) Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolated in Cambodia between 2016 and 2020. METHODS: E. coli (n = 223) and K. pneumoniae (n = 39) were tested for colistin minimum inhibitory concentration (MIC) by broth microdilution. Resistant isolates were subjected to polymerase chain reaction (PCR) for detection of mobile colistin resistance genes (mcr) and chromosomal mutations in the two-component system (TCS). RESULTS: Eighteen isolates (10 K. pneumoniae and 8 E. coli) revealed colistin resistance with a rate of 5.9% in E. coli and 34.8% in K. pneumoniae among ESBL isolates, and 1% in E. coli and 12.5% in K. pneumoniae among CP isolates. The resistance was associated with mcr variants (13/18 isolates, mcr-1, mcr-3, and mcr-8.2) and TCS mutations within E. coli and K. pneumoniae, with the first detection of mcr-8.2 in Cambodia, the discovery of new mutations potentially associated to colistin resistance in the TCS of E. coli (PhoP I47V, PhoQ N352K, PmrB G19R, and PmrD G85R) and the co-occurrence of mcr genes and colistin resistance conferring TCS mutations in 11 of 18 isolates. CONCLUSIONS: The findings highlight the presence of colistin resistance in ESBL- and CP- Enterobacteriaceae involved in human infections in Cambodia as well as chromosomal mutations in TCS and the emergence of mcr-8.2 in E. coli and K. pneumoniae. It underscores the need for continuous surveillance, antimicrobial stewardship, and control measures to mitigate the spread of colistin resistance.

Efficient disinfection of combined sewer overflows by ultraviolet/peracetic acid through intracellular oxidation with preserving cell integrity.

Combined sewer overflows (CSOs) introduce microbial contaminants into the receiving water bodies, thereby posing risks to public health. This study systematically investigated the disinfection performance and mechanisms of the combined process of ultraviolet and peracetic acid (UV/PAA) in CSOs with selecting Escherichia coli (E. coli) as a target microbial contaminant. The UV/PAA process exhibited superior performance in inactivating E. coli in simulated CSOs compared with UV, PAA, and UV/H2O2 processes. Increasing the PAA dosage greatly enhanced the disinfection efficiency, while turbidity and organic matter hindered the inactivation performance. Singlet oxygen (1O2), hydroxyl (•OH) and organic radicals (RO•) contributed to the inactivation of E. coli, with •OH and RO• playing the prominent role. Variations of intracellular reactive oxygen species, malondialdehyde, enzymes activities, DNA contents and biochemical compositions of E. coli cells suggested that UV/PAA primarily caused oxidative damage to intracellular molecules rather than the damage to the lipids of the cell membrane, therefore effectively limited the regrowth of E. coli. Additionally, the UV/PAA process displayed an outstanding performance in disinfecting actual raw CSOs, achieving a 2.90-log inactivation of total bacteria after reaction for 4 min. These results highlighted the practical applicability and effectiveness of the UV/PAA process in the disinfection of CSOs.

Cryo-EM structure of cytochrome bo3 quinol oxidase assembled in peptidiscs reveals an "open" conformation for potential ubiquinone-8 release.

Cytochrome bo3 quinol oxidase belongs to the heme­copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1-41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a "U-shape" assembly in contrast to the transmembrane helix, named "TM0", in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo3 conformations should exist. Our structural model presents experimental evidence for an "open" conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.

Integrating quantitative microbiological risk assessment and disability-adjusted life years to evaluate the effects of urbanization on health risks for river recreationists.

Urban rivers provide an excellent opportunity for water recreation. This study probabilistically assessed health risks associated with water recreation in urban rivers in the Bitan Scenic Area, Taiwan, by employing quantitative microbial risk assessment and disability-adjusted life years (DALYs). Moreover, the effects of urbanization on the health risks of river recreation induced by waterborne pathogenic Escherichia coli (E. coli) were investigated. First, data on river E. coli levels were collected in both the Bitan Scenic Area and the upstream river section, and model parameters were obtained through a questionnaire administered to river recreationists. Monte Carlo simulation was then employed to address parameter uncertainty. Finally, DALYs were calculated to quantify the cumulative effects in terms of potential life lost and years lived with disability. The results indicated that the 90 % confidence intervals for the disease burden (DB) were 0.2-74.1 × 10-6, 0.01-94.0 × 10-6, and 0.3-128.9 × 10-6 DALY per person per year (pppy) for canoeing, swimming, and fishing, respectively, in the Bitan Scenic Area. Furthermore, urbanization near the Bitan Scenic Area approximately doubled the DB risks to river recreationists in upstream rural areas. At the 95th percentile, the DB risks exceeded the tolerances recommended by the World Health Organization (1 × 10-6) or U.S. Environmental Protection Agency (1 × 10-4). The findings suggest that the simultaneous implementation of effluent sewer systems and best management practices can reduce health risks to river recreationists by at least half, reducing the DALY levels below 1 × 10-4 or even 1 × 10-5 pppy.

Distance-based paper analytical devices integrated with molecular imprinted polymers for Escherichia coli quantification.

The development of distance-based paper analytical devices (dPADs) integrated with molecularly imprinted polymers (MIPs) to monitor Escherichia coli (E. coli) levels in food samples is presented. The fluidic workflow on the device is controlled using a designed hydrophilic bridge valve. Dopamine serves as a monomer for the formation of the E. coli-selective MIP layer on the dPADs. The detection principle relies on the inhibition of the E. coli toward copper (II) (Cu2+)-triggered oxidation of o-phenylenediamine (OPD) on the paper substrate. Quantitative detection is simply determined through visual observation of the residual yellow color of the OPD in the detection zone, which is proportional to E. coli concentration. The sensing exhibits a linear range from 25.0 to 1200.0 CFU mL-1 (R2 = 0.9992) and a detection limit (LOD) of 25.0 CFU mL-1 for E. coli detection. Additionally, the technique is highly selective with no interference even from the molecules that have shown to react with OPD to form oxidized OPD. The developed device demonstrates accuracy and precision for E. coli quantification in food samples with recovery percentages between 98.3 and 104.7% and the highest relative standard deviation (RSD) of 4.55%. T-test validation shows no significant difference in E. coli concentration measured between our method and a commercial assay. The proposed dPAD sensor has the potential for selective and affordable E. coli determination  in food samples without requiring sample preparation. Furthermore, this strategy can be extended to monitor other molecules for which MIP can be developed and integrated into paper-microfluidic platform.

Molecular Characterization of Escherichia coli Causing Urinary Tract Infections Through Next-Generation Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes.

Introduction An enormous increase in antimicrobial resistance (AMR) among bacteria isolated from human clinical specimens contributed to treatment failures. Increased surveillance through next-generation sequencing (NGS) or whole genome sequencing (WGS) could facilitate the study of the epidemiology of drug-resistant bacterial strains, resistance genes, and other virulence determinants they are potentially carrying. Methods This study included 30 Escherichia coli (E. coli) isolates obtained from patients suffering from urinary tract infections (UTIs) attending Prathima Institute of Medical Sciences, Karimnagar, India. All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS to identify genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes carried by the isolates. Results The conventional antimicrobial susceptibility testing revealed that 15 (50%) isolates were resistant to imipenem (IPM), 10 (33.33%) were resistant to amikacin (AK), 13 (43.33%) were resistant to piperacillin-tazobactam (PTZ), 17 (56.66%) were resistant to cephalosporins, and 14 (46.66%) were resistant to nitrofurantoin (NIT). Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaCTX-M (19/30, 63.33%) genes, followed by blaTEM and blaOXA-1. The blaNDM-5 gene was found in three isolates (3/30, 10%). The virulence genes identified in the present study were iutA, sat, iss, and papC, among others. The E. coli serotype found predominantly belonged to O25:H4 (5, 16.66%), followed by O102:H6 (4, 13.33%). A total of 16 MLST variants were identified among the examined samples. Of the MLST-based sequence types (STs) identified, ST-131 (7, 23.33%) was the predominant one, followed by ST-167 (3, 10%) and ST-12 (3, 10%). Conclusions The study results demonstrated that the E. coli strains isolated from patients suffering from UTIs potentially carried antimicrobial resistance and virulence genes and belonged to different strain types based on MLST. Careful evaluation of bacterial strains using molecular analyses such as NGS could facilitate an improved understanding of bacterial antibiotic resistance and its virulence potential. This could enable physicians to choose appropriate antimicrobial agents and contribute to better patient management, thereby preventing the emergence and spread of drug-resistant bacteria.

Extended-Spectrum Beta-Lactamase Escherichia coli-Associated Acute Cholangitis: Uncommon Patient Characteristics and Clinical Implications.

Acute cholangitis is a potentially life-threatening condition caused by an infection of the biliary tract resulting from biliary obstruction. This case report highlights an unusual presentation of acute cholangitis in an elderly patient characterized by the presence of extended-spectrum beta-lactamase-producing Escherichia coli. We aim to emphasize the significance of recognizing diverse clinical manifestations in the elderly population to enhance timely diagnosis and appropriate management. The case highlights the importance of better understanding patient risk factors for potential causative organisms and their susceptibility to selecting proper antibiotics and improving clinical outcomes.

Environmental predictors of Escherichia coli concentration at marine beaches in Vancouver, Canada: a Bayesian mixed-effects modelling analysis.

Understanding historical environmental determinants associated with the risk of elevated marine water contamination could enhance monitoring marine beaches in a Canadian setting, which can also inform predictive marine water quality models and ongoing climate change preparedness efforts. This study aimed to assess the combination of environmental factors that best predicts Escherichia coli (E. coli) concentration at public beaches in Metro Vancouver, British Columbia, by combining the region's microbial water quality data and publicly available environmental data from 2013 to 2021. We developed a Bayesian log-normal mixed-effects regression model to evaluate predictors of geometric E. coli concentrations at 15 beaches in the Metro Vancouver Region. We identified that higher levels of geometric mean E. coli levels were predicted by higher previous sample day E. coli concentrations, higher rainfall in the preceding 48 h, and higher 24-h average air temperature at the median or higher levels of the 24-h mean ultraviolet (UV) index. In contrast, higher levels of mean salinity were predicted to result in lower levels of E. coli. Finally, we determined that the average effects of the predictors varied highly by beach. Our findings could form the basis for building real-time predictive marine water quality models to enable more timely beach management decision-making.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

A novel strategy for specific sensing and inactivation of Escherichia coli: Constructing a targeted sandwich-type biosensor with multiple SERS hotspots to enhance SERS detection sensitivity and near-infrared light-triggered photothermal sterilization performance.

Human health is greatly threatened by bacterial infection, which raises the risk of serious illness and death in humans. For early screening and accurate treatment of bacterial infection, there is a strong desire to undertake ultrasensitive detection and effective killing of pathogenic bacteria. Herein, a novel surface-enhanced Raman scattering (SERS) biosensor based on sandwich structure consisting of capture probes/bacteria/SERS tags was established for specific identification, capture and photothermal killing of Escherichia coli (E. coli). Finite-difference time-domain (FDTD) technique was used to simulate the electromagnetic field distribution of capture probes, SERS tags and sandwich-type SERS substrate, and a possible SERS enhancement mechanism based on sandwich structure was presented and discussed. Sandwich-type SERS biosensor successfully achieved distinctive identification and magnetic beneficiation of E. coli. In addition, a single SERS substrate, including capture probes and SERS tags, could also achieve outstanding photothermal effects as a consequence of localized surface plasmon resonance (LSPR) effect. Intriguingly, sandwich-type SERS biosensor demonstrated a higher photothermal conversion efficiency (50.03 %) than the single substrate, which might be attributed to the formation of target bacterial clusters. The superior biocompatibility and the low toxicity of the sandwich-type biosensor were confirmed. Our approach offers a fresh method for constructing sandwich-type biosensor with multiple SERS hotspots based on extremely effective hybrid plasmonic nanoparticles, and has a wide range of potential applications in the recognition and treatment of bacteria.

Development and verification of an ELISA method for detection of process-specific residual host protein in biological preparation / 中国生物制品学杂志

@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.

The genetic network underlying the evolution of pathogenicity in avian Escherichia coli.

Introduction: Colibacillosis is a worldwide prevalent disease in poultry production linked to Escherichia coli strains that belong to the avian pathogenic E. coli (APEC) pathotype. While many virulence factors have been linked to APEC isolates, no single gene or set of genes has been found to be exclusively associated with the pathotype. Moreover, a comprehensive description of the biological processes linked to APEC pathogenicity is currently lacking. Methods: In this study, we compiled a dataset of 2015 high-quality avian E. coli genomes from pathogenic and commensal isolates, based on publications from 2000 to 2021. We then conducted a genome-wide association study (GWAS) and integrated candidate gene identification with available protein-protein interaction data to decipher the genetic network underlying the biological processes connected to APEC pathogenicity. Results: Our GWAS identified variations in gene content for 13 genes and SNPs in 3 different genes associated with APEC isolates, suggesting both gene-level and SNP-level variations contribute to APEC pathogenicity. Integrating protein-protein interaction data, we found that 15 of these genes clustered in the same genetic network, suggesting the pathogenicity of APEC might be due to the interplay of different regulated pathways. We also found novel candidate genes including an uncharacterized multi-pass membrane protein (yciC) and the outer membrane porin (ompD) as linked to APEC isolates. Discussion: Our findings suggest that convergent pathways related to nutrient uptake from host cells and defense from host immune system play a major role in APEC pathogenicity. In addition, the dataset curated in this study represents a comprehensive historical genomic collection of avian E. coli isolates and constitutes a valuable resource for their comparative genomics investigations.

Superhydrophobic Rotation-Chip for Computer-Vision Identification of Drug-Resistant Bacteria.

The transport, distribution, and mixing of microfluidics often require additional instruments, such as pumps and valves, which are not feasible when operated in point-of-care (POC) settings. Here, we present a simple microfluidic pathogen detection system known as Rotation-Chip that transfers the reagents between wells by manually rotating two concentric layers without using external instruments. The Rotation-Chip is fabricated by a simple computer numerical control (CNC) machining process and is capable of carrying out 60 multiplexed reactions with a simple 30 or 60° rotation. Leveraging superhydrophobic coating, a high fluid transport efficiency of 92.78% is achieved without observable leaking. Integrated with an intracellular fluorescence assay, an on-chip detection limit of 1.8 × 106 CFU/mL is achieved for ampicillin-resistant Escherichia coli (E. coli), which is similar to our off-chip results. We also develop a computer vision method to automatically distinguish positive and negative samples on the chip, showing 100% accuracy. Our Rotation-Chip is simple, low-cost, high-throughput, and can display test results with a single chip image, making it ideal for various multiplexing POC applications in resource-limited settings.

High-throughput, highly sensitive and rapid SERS detection of Escherichia coli O157:H7 using aptamer-modified Au@macroporous silica magnetic photonic microsphere array.

The aim of this research was to develop a simple, rapid, sensitive, high-throughput detection method for foodborne Escherichia coli (E. coli) O157:H7 based on the aptamer-modified gold nanoparticles@macroporous magnetic silica photonic microsphere (Au@MMSPM). Such Au@MMSPM array system for E. coli O157:H7 not only integrated sample pretreatment with rapid detection, but also showed highly enhanced effect to develop a highly sensitive SERS assay. The established SERS assay platform gave a wide linear detection range (10-106 CFU/mL) and low limit of detection (2.20 CFU/mL) for E. coli O157:H7. The whole analysis time including sample pretreatment and detection was 110 min. This SERS-based assay platform provided a new high-throughput, highly sensitive and fast detection technology for monitoring E. coli O157:H7 in real samples from the fields of food industry, medicine and environment.

Genotypic and Phenotypic Characterization of Escherichia coli Isolates Recovered from the Uterus of Mares with Fertility Problems.

Escherichia coli is the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24 E. coli strains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%) E. coli strains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, with fimH detected in all strains, and followed by kpsMTII (11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on plates versus strains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight into E. coli strains associated with mares' infertility. These results broaden the knowledge of E. coli and, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares.

Categorization of Escherichia coli outer membrane proteins by dependence on accessory proteins of the ß-barrel assembly machinery complex.

The outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique ß-barrel transmembrane domain. Most OMPs are assembled into the OM by the ß-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics.